ABSTRACT In this application we will perform high throughput, high content, screening for small molecules that improve kidney regeneration after acute kidney injury (AKI). AKI presents a dire unmet medical need because of its high prevalence with long-term adverse health effects and life-threatening sequelae. Mortality is high and the only effective treatments are renal replacement therapies. The onset of the precipitating event is unpredictable, and in many instances once patients are admitted to the hospital, injury has already occurred. Improving recovery from injury therefore presents an attractive opportunity for intervention, but to date, no therapies are available that are effective if administered post injury. The vertebrate kidney has an innate ability to regenerate and follows a well-defined cellular mechanism that encompasses dedifferentiation of surviving renal tubule cells, proliferation of resulting progenitors, and repopulation of the denuded tubule. This sequence of events, together with their respective molecular markers, is conserved between humans, mouse, and zebrafish. During regeneration, transcription factors normally expressed during organogenesis (e.g.,lhx1a, pax2, and pax8) are reactivated. We previously demonstrated that small molecule-mediated augmentation of endogenous Lhx1a expression can ameliorate recovery in zebrafish and mouse models of AKI. Together these data support the overall hypothesis that augmentation by small molecules of cellular programs that drive kidney repair after injury represents a novel pharmacologic approach for the treatment of AKI and associated sequelae. Using a transgenic zebrafish line that expresses Lhx1a-EGFP we have developed an artificial intelligence-based, high-content assay to quantify lhx1a expression in the living embryo. Using multivariate analysis, the assay met accepted HTS assay performance standards and was validated in three-day variability studies and a small pilot library screen. We will perform a primary HTS of 50,000 compounds from the MLPCN collection. Prioritized hits will be subjected to a fully implemented, rigorous secondary assay paradigm encompassing kidney organ development, metabolic stability, in vivo efficacy, and activity profiling in a pathophysiological relevant AKI model. At the end of these studies we will have identified functionally and mechanistically characterized in vivo chemical probes to investigate the biology of kidney injury and regeneration, some of which are expected to have features that make them suitable for development into preclinical leads.